2012 September 26
Matters arising Are students familiar with DNA analysis facility? Should we arrange a field trip? Remaining YFG presentations #Rachael- YFG presentation #Lisz- YFG presentation (if she's healthy) Minilecture (JMB): Development of DNA sequencing methods Class Notes History of DNA Sequencing ''' '''Proteins: The first sequenced protein was Insulin and was sequenced by Fred Sanger. Proteins were sequenced prior to RNA & DNA because there were better analytical methods for proteins, they are simple to extract and purify due in part to their solubility in water and because proteins are made from 20 amino acids (each having unique chemical properties) which are easy to isolate from each other. Nucleic Acids: The first sequenced nucleic acid was mitochondrial DNA. This was a clear choice because it has one circular chromosome that could be easily isolated. Nucleic acids were initially difficult to isolate and purify because it they only contain 4 nucleotides (A, T, C, G) that are all chemically similar and in organisms with diploid genomes, individual chromosomes were difficult to isolate. Availability of technology was also an obstacle because molecular cloning and PCR needed to be invented prior to any nucleic acid sequencing. RNA: tRNA was first sequenced in the 1960's & 70's. tRNA was the first RNA to be sequenced because it was easy to isolate, small, abundant, unique chemically (ie. each had its own amino acid attached) and tRNA's contain modified nucleotides. The tRNA could be isolated and then fractionated similar to protein purification schemes to separate one tRNA from another tRNA. DNA: DNA isolation required the isolation of individual DNA fragments. Two methods were then developed to sequence the DNA. Method 1: the Sanger Dideoxy Method, which is still used today. Method 2: Maxam-Gilbert Chemical Modification Method. Using the Maxam-Gilbert Chemical Modification Method, the DNA molecule is chemically labeled with a radioactive isotope at one end of the DNA. The DNA is subjected to various chemical reactions and then a cleavage reaction. The resulting fragments are separated by gel electrophoresis and the sequence is then determined. The Sanger method is an enzymatic reaction. It takes advantage of one particular modified nucleotide - dideoxynucleotide (ddNTP). During DNA replication (by using the same primers), the DNA polymerase incorporates the ddNTP, but the replication process cannot continue further on. The fragments are then separated on a gel. This proces is nowadays automated, by using radioactivity (but modifications to the procedure include replacing isotopes with fluorescent dyes (red, blue, green, yellow). This method is still used and is highly accurate with reasonably long read lengths. (50-150 nucleotides) Today technology has brought next generation sequencers (also called massively parallel sequencing). No DNA cloning or PCR is required. De-novo sequencing: sequencing of a new genome of a new organism. It is necessary to collect a lot of data and put them together. This allows you to indentify problem areas. Because high-throughput sequencing gives only short reads (50-150ntd), problem areas are usually filled in by Sanger sequencing, which allows long reads. There are also rumors of a new method by the company Oxford Nanopore. Assignments for Mon 1 October #Chapter 3 exercises, problems and weblems #Add content to the following articles on DNA sequencing: | DNAseq: 454 pyrosequencing | DNAseq: Ion Torrent | DNAseq: Maxam-Gilbert | DNAseq: Oxford Nanopore | DNAseq: Sanger dideoxy | DNAseq: Illumina | DNAseq: SOLiD |